The present invention generally relates to modification of the adenovirus fiber protein and methods for use thereof to modify cellular attachment by the fiber protein.
In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis is reviewed by Brown and Greene, DNA and Cell Biology 1991 10:6, 399-409. A number of viruses infect cells via a receptor-ligand interaction. Adenovirus is an example of a virus that utilizes receptor-mediated endocytosis to internalize infectious virus.
Gene therapy requires transfer of recombinant nucleic acid constructs into cells. Although a number of different methods for gene transfer have been proposed, one of the most promising remains by utilizing recombinant viruses. The development of recombinant adenoviruses for this purpose has had a number of applications, based upon the unique advantages of this system.
One advantage is that recombinant adenoviruses have been isolated and characterized that contain genomic deletions that render the virus replication incompetent except within cell lines that trans-complement the deleted functions. The cell lines that contain the viral genes required for production of infective viral particles are called packaging cell lines. The construction of the replication-defective adenoviruses is described by Berkner et al., 1987 J. Virology 61:1213-1220 (1987); Massie et al., 1986 Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., 1986 J. Virology 57:267-274 (1986); Davidson et al., 1987 J. Virology 61:1226-1239 (1987); Zhang xe2x80x9cGeneration and identification of recombinant adenovirus by liposome-mediated transfection and PCR analysisxe2x80x9d BioTechniques 1993 15:868-872. The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infectious viral particles.
The ability of the virus to achieve high levels of expression of therapeutic gene products, and the capacity of the virus to infect non-dividing, terminally differentiated cells, has been exploited for gene therapy applications requiring direct, in vivo gene delivery. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites, as reported by Morsy, 1993 J. Clin. Invest. 92:1580-1586; Kirshenbaum, 1993 J. Clin. Invest. 92:381-387; Roessler, 1993 J. Clin. Invest. 92:1085-1092; Moullier, 1993 Nature Genetics 4:154-159; La Salle, 1993 Science 259:988-990; Gomez-Foix, 1992 J. Biol. Chem. 267:25129-25134; Rich, 1993 Human Gene Therapy 4:461-476; Zabner, 1994 Nature Genetics 6:75-83; Guzman, 1993 Circulation Research 73:1201-1207; Bout, 1994 Human Gene Therapy 5:3-10; Zabner, 1993 Cell 75:207-216; Caillaud, 1993 Eur. J. Neuroscience 5:1287-1291; and Ragot, 1993 J. Gen. Virology 74:501-507.
Whereas the broad tropism of the adenovirus has allowed transduction of a variety of tissue types for certain gene therapy applications, this broad host range may also create additional problems. It is generally recognized that in the context of direct in vivo gene delivery, the ability to target heterologous genes to specific cellular targets would be highly advantageous, as reviewed by Anderson, 1992 Science 256:808-813; McCabe, 1993 Biochemical Medicine and Metabolic Biology 50:241-253. The promiscuous tropism of the adenovirus could potentially undermine those strategies where cell-specific delivery is essential. In this regard, it has been shown that systematically administered adenovirus transduces multiple tissue types (Stratford-Perricaudet, 1992 J. Clin. Invest. 90:626-630). This nonspecific delivery could be potentially deleterious, since ectopic expression of transferred, heterologous genes could occur in inappropriate cellular targets as a result. Furthermore, the ubiquitous binding of the virus may require systemic administration of prohibitive doses of virus in order to adequate transduce a specific target site in vivo.
In addition to the binding of a broad virus tropism in vivo, the lack of adenovirus binding to a given target cell may obviate the utility of this vector for some gene therapy applications. For example, for mature differentiated muscle cells, the lack of a receptor for adenovirus prevents use of adenoviral gene transfer strategies for targeting this tissue type, as reviewed by Ragot, 1993; and Karpati, 1993 Muscle and Nerve 16:1141-1153). Thus, it would be useful in certain instances to broaden the tropism of recombinant adenovirus to allow gene delivery to selected cell subsets not presently transducible with this vector system.
Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus, as reported by Chardonnet and Dales, 1970 Virology 40:462-477; Brown and Burlingham, 1973 J. Virology 12:386-396; Svensson and Persson, 1985 J. Virology 55:442-449; Seth, et al., 1984 J. Virol. 51:650-655; Seth, et al., Mol. Cell. Biol. 1984 4:1528-1533; Varga et al., 1991 J. Virology 65:6061-6070 (1991); Wickham et al., 1993 Cell 73:309-319). The adenovirus binds to the receptor via the knob portion of the fiber protein that projects outward from the viral coat. The fiber protein is actually a homotrimeric protein encoded by the fiber gene within the adenoviral genome. Adenovirus capsid proteins, which include the hexon, penton base, and fiber proteins, are synthesized late in infection in the cytoplasm and transported to the nucleus for assembly into virus particles.
Fiber plays a crucial role in adenovirus infection by attaching the virus to a specific receptor on the cell surface. The fiber consists of three domains: an N-terminal tail that interacts with penton base, a shaft composed of 22 repeats of a 15 amino acid segment that forms xcex2-sheet and xcex2-bends, and a knob at the C-terminus that contains the type-specific antigen and is responsible for binding to the cell surface receptor. The fiber protein is also responsible for transport of viral nucleic acids into the nucleus. The gene encoding the fiber protein from adenovirus serotype 2 has been expressed in human cells and shown by using a recombinant vaccinia virus vector to be correctly assembled into trimers, glycosylated and transported to the nucleus, as reported by Hong and Engler, 1991 Virology 185, 758-767.
Alteration of gene delivery mediated by recombinant adenovirus to specific cell types would have great utility for a variety of gene therapy applications.
It is therefore an object of the present invention to provide a method and means by which adenovirus can be targeted to infect specific cell types.
It is a further object of the present invention to provide a method and means by which adenovirus proteins can be used to target nucleic acid or protein delivery to a specific cell or the nucleus of a specific cell.
The fiber protein of adenovirus has been genetically altered via attachment at the carboxyl end of a peptide linker, preferably up to 26 amino acids in length which forms a random coil, which can be used to attach a non-adenovirus ligand altering the binding specificity of the fiber protein. Examples of ligands include peptides which are selectively bound by a targeted cell so that the modified fiber protein is internalized by receptor-mediated endocytosis, and peptides which can act as an universal coupling agent, for example, biotin or strepavidin. The linker is designed to not interfere with normal trimerization of fiber protein, to avoid steric hindrance of binding of the fiber protein to a targeted cell, and to serve as a site to introduce new peptide sequence. The modified fiber protein is prepared by genetic engineering of the nucleotide sequence encoding the fiber protein, through the addition of new sequence at the carboxyl tail-encoding region which encodes the linker and the ligand. The N-terminus of the fiber protein is not altered in the preferred embodiment, although in some embodiments it may be desirable to inhibit uptake by the nucleus of the fiber protein, by deletion of nuclear targeting signals.
The modified fiber protein can be utilized as part of a recombinant adenovirus for use in gene therapy.